The present invention relates to analytical instruments and, more particularly, to flow particle analysis such as used in cytometry. A major objective of the present invention is to reduce false triggering in multi-laser flow cytometry systems.
A typical single-laser flow cytometry system includes a flow subsystem for flowing a particle stream along a flow path, a blue laser illumination source for illuminating a location along the flow path, several photodetectors for detecting light modulations (e.g., due to emissions, absorption, and scattering) at the illuminated flow path location, analog pulse-processing circuitry for processing the photodetector outputs, a digitizer, and a digital data processor (computer) for analyzing digitized detector data to characterize the particle stream.
The photodetectors can include a detector for forward-scattered light, a detector for side-scattered light, a green fluorescence detector, a yellow fluorescence detector, and a red fluorescence detector. The scatter detectors can be used to detect the presence and indicate the size of a particle at the illuminated location. The fluorescence detectors can be used to distinguish three fluorochromes: a blue-excited green fluorochrome (e.g., FITC), a blue-excited yellow fluorochrome (e.g., RPE), and a blue-excited red fluorochrome (e.g., PerCP).
Attempts to provide for four-way fluorochrome distinctions using a single-laser system have proved difficult due to ambiguities caused by overlap of emissions spectra. Improved four-way fluorochrome distinction has been achieved using dual-laser systems. A typical dual-laser system adds a red laser illumination source to illuminate a second location along the flow path. The additional laser allows a red-excited (and not blue-excited) red fluorochrome (e.g., APC) to be distinguished from a blue-excited (and not red-excited) red fluorochrome (e.g., PerCP). Each of these red fluorochromes are readily distinguished from the blue-excited green fluorochrome and the blue-excited yellow fluorochrome. Research and development is ongoing on extending the multi-laser approach to provide for greater than four-way fluorochrome distinctions.
The photodetector outputs not only serve as the sources of data to be analyzed, but also determine what portions of the data are to be analyzed. To this end, one or a combination of photodetector outputs can be used to derive a trigger signal that activates processing, including digitization. In the absence of a trigger signal, signals from the photodetector outputs are gated. Peak and hold circuits and subsequent digitizers are examples of analog components that can require recovery time after processing a signal. Therefore, limiting amplification of uninteresting signals can help ensure appropriate processing of interesting data. Furthermore, gating limits the amount of data that must be digitized, processed and stored.
In general, a trigger determines a window of time within which interesting data is expected and, therefore, data processing is activated. Often, a trigger is initiated when a scatter detector output crosses a threshold--indicating with a high level of certainty that a particle is in the flow-path location corresponding to the detector. The fluorescent particle detector outputs can then be amplified, digitized, and processed to identify any fluorochromes in the particle--so that the particle (which may be inherently fluorescent or tagged with one or more fluorescent markers) can be characterized.
Accurate triggering is important. A false negative, i.e., no trigger when a particle (or alternative indicator of interesting data) is present, causes a loss of valuable data. A false positive, a trigger when no particle or alternative indicator of interesting data is present, burdens the analog and digital processing systems. Furthermore, many flow analyzers fail to process new events while processing data relating to the supposed event associated with a trigger. During this "dead time", interesting events can avoid detection, causing a loss of interesting data. Thus, both false positives and false negatives can cause a loss of data required for an accurate analysis. It should be noted that the data loss is often systematic in that certain types of interesting events are more likely to be discarded than others. Thus false triggering can bias the analysis.
In a single-laser system, processing dead time after a trigger can be on the order of the time it takes a particle to traverse the laser beam where it intersects the flow path; this may be a couple of microseconds. In a dual-laser system, the processing dead time can be on the order of the transit time a particle takes in moving from the upstream detection location to the downstream detection location; this may be seven to twenty-five microseconds. More generally, in a multi-laser system, the dead time is on the order of the transit time between the first and last detection locations. Thus, the penalty in lost interesting data for false positive triggers in a dual- or other multi-laser system can be substantial.
Processing dead time in a multi-laser system can be reduced by minimizing the separation of the illumination locations. The cost of this solution is increased interlocation emissions crosstalk between illumination locations. In other words, light emitted or scattered at a first illumination location can leak to a second location so that it is detected by photodetectors arranged to detect light at the second location. In particular, red-excited red fluorescence can be detected by a detector arranged to detect blue-excited red fluorescence. This can lead to a false indication of a blue-excited red fluorochrome when there is no such material at the location illuminated by the blue laser. The converse false indication of a red-excited red fluorochrome at the flow-path location illuminated by the blue laser can also occur.
In the case a particle stimulates fluorescent emission at a location upstream of the location indicated by the detection circuitry, the false indication is referred to as a "pre-pulse". In the case a particle stimulates fluorescent emission at a location downstream of the location indicated by the detection circuitry, the false indication is referred to as a "post-pulse". Herein, the term "shadow pulse" is used to encompass both pre-pulses and post-pulses.
In general, a shadow pulse associated with a trigger signal is outside of the trigger window. For example, in a dual-laser system that triggers on the upstream illumination location, the output of a detector for the downstream illumination location is not processed until the triggering particle has had time to reach the downstream location. The pre-pulse generated concurrently with the trigger is ignored.
However, a trigger particle might fail to trigger if it passes the upstream trigger location during a dead time. If it causes a strong modulation of light when it reaches the downstream illumination location, the resulting post-pulse could result in a trigger. Since the downstream photodetector output would be ignored, there would be no indication that the trigger was false. Hence, a penalty in processing activity and dead time would ensue with no offsetting acquisition of useful data. In fact, there would be a false indication of a non-fluorescent particle; such "selective elimination" would distort the determination of the sample composition.
Evaluating the lymphocyte content of a blood sample is an example of an application in which a shadow pulse could induce a false trigger. The blood sample can be lysed and unwashed, so that sample is filled with uninteresting particles, i.e., debris. The debris particles can be so numerous relative to the white cells of interest that particle presence is not a sufficiently selective criterion for triggering. In fact, the debris can be sufficiently dense that scattering detections are practically continuous, rendering the scatter detectors essentially useless for such a sample.
Accordingly, it is appropriate to tag white cells with a fluorochrome that can be used as a trigger. From a sample preparation standpoint, it can be most convenient to tag white blood cells with blue-excited red fluorochrome, e.g., PerCP, which thus becomes the triggering fluorochrome. Other fluorochrome tags are attached to specific respective lymphocytes.
Fluorochrome detections tend to be much weaker than scatter detections; as a result, trigger thresholds for fluorochrome triggering must be set near the noise floor for the respective fluorochrome detector. A (e.g., blue-excited-red indication) shadow pulse at the trigger location, resulting from a (e.g., red-excited red) fluorochrome at the non-trigger location, can be sufficient to cross the trigger threshold at the location.
The options appear to be to accept the false triggering, to avoid samples where such false triggering is likely to arise, or to develop a scheme to minimize such false triggering where it is otherwise likely to arise. Clearly, the last option is the most attractive.